Mechanisms regulating the responsiveness of cells to substance P: Cell-surface degradation of substance P and NK-1 receptor endocytosis

A. M. Garland, E. F. Grady, D. G. Payan, N. W. Bunnett

Research output: Contribution to journalArticle

Abstract

Substance P (SP) degradation by the cell-surface enzyme neutral endopeptidase (NEP), and endocytosis of the NK-1 receptor (NK-1R) are potential mechanisms that limit the responsiveness of cells to SP. These mechanisms were studied in epithelial cells (KNRK) transfected with cDNA encoding an epitope labeled Flag-NK-1R or NEP. In cells expressing NK-1R alone, specific saturable 125I-Sp binding at 37°C was 28% ± 3% of total counts. Co-expression of NEP with the NK-1R reduced binding to 4 ± 0.3% of total counts. Addition of the NEP inhibitor thiorphan restored binding to control levels. In cells expressing NK-1R alone, SP (1 nM) stimulated an increase in [Ca2+](i) of 163 ± 16 nM. Go-expression of NEP reduced the [Ca2+](i) response to 52 ± 13 nM, and this response was restored to control levels by thiorphan. Antibodies to the NK-1R and rhodamine labeled SP were used to observe endocytosis in KNRK cells expressing the NK-1R alone. When cells were incubated with SP for 60 min at 4°C, NK-1R and rhodamine SP were confined to the plasma membrane. After 3 and 10 min at 37°C, there was a reduction in cell-surface staining and NK-1R and rhodamine-SP were localized in intracellular vesicles. Binding experiments with 125I-Sp confirmed the rapid internalization of peptide. Therefore, the NK-1R and SP are internalized within minutes of binding. Thus, ligand degradation and receptor endocytosis limit the cellular response to SP.

Original languageEnglish (US)
Pages (from-to)5-13
Number of pages9
JournalBiomedical Research
Volume16
Issue numberSUPPL. 2
StatePublished - 1995

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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